Abstract Diversity of the T cell receptor is mainly developed through V(D)J rearrangement and N addition during TCR development. The product of V(D)J rearrangement is the CDRβ3, a region of high variability that recognizes antigen and includes all of the D gene. The Dβ sequence is highly conserved across various species, from trout to mouse to humans. This suggests that there are some natural constraints on the TCR; these constraints are thought to limit deleterious T cells from reaching the periphery. We hypothesize that altering the D region will have an effect on the development of thymocytes. To do this we have created Dβ altered mice. These mutants are a Dβ2 KO (Dβ1); a replacement of the Dβ locus with a charged Dβ (DβDKRQ); and a replacement of the Dβ locus with a hydrophobic DH (DβYTL). When compared to WT mice, the mutant Dβ mice have an altered T cell repertoire in both CDRβ3 amino acid composition and length and these differences can be attributed to the changes in the germline sequence. Changing the Dβ also changes the total T cell number in both developing and mature T cells, with an altered Dβ being selected against. However, Treg numbers from the DβDKRQ mice are increased in the mucosa, suggesting a link between charge, self-reactivity and nTreg induction. Ongoing experiments will elucidate the role of the Dβ sequence on the development of T cells and the role it plays in infection and inflammation.